Pulmonary inflammation is characterized by plasma exudation into the airspace and the subsequent formation of a fibrin clot. The persistence of this protein-rich matrix in the airway may lead to mesenchymal cell ingrowth and obliterative scarification. Effective mechanisms for clearing luminal fibrin are therefore required to restore normal pulmonary architecture and function following injury. Pulmonary epithelial cells can mediate the lysis of fibrin clots by mechanisms that require urokinase- dependent plasminogen activation at the cell surface. This plasminogen activating activity is focused to the plasma membrane by a high affinity binding protein, the urokinase receptor (uPAR). To better understand epithelial-directed repair processes in the human airway, this proposal will study urokinase receptor expression utilizing primary cultures of airway epithelial cells as a model. A unique feature of this application is its comprehensive approach designed to study many facets of airway epithelial cell uPAR expression. To direct fibrinolytic activity to the airway lumen, the uPAR should be preferentially expressed on the apical plasma membrane. To test this hypothesis, immunofluorescent Confocal microscopy will be used to localize uPAR on the epithelial cell. The effects of cell spreading during monolayer healing on uPAR surface expression will also be examined. Preliminary studies reveal that airway epithelial cell fibrinolytic activity is downregulated by dexamethasone suggesting that corticosteroids might have a detrimental effect on lung healing. A detailed study of this response will be made using measurements of uPAR mRNA synthesis, protein kinetics, and the functional correlates of uPAR expression, cell surface urokinase binding and fibrinolytic activity. Investigations into the effects of transforming growth factor beta on airway epithelial uPAR expression will provide information relevant to the importance of profibrogenic cytokines in pulmonary repair processes. The critical role for uPAR in cell-mediated fibrinolysis will be demonstrated using specific inhibitors of urokinase binding. Additional investigations into the importance of surface bound uPA in fibrinolysis will utilize uPAR gene transfection to engender a fibrinolytic "null cell" with the ability to lyse plasma clots. Finally, the tools and observations developed during the course of this project will be applied to the study of uPAR expression in tissue samples from patients with inflammatory lung disease. Overall, this proposal outlines a multidisciplinary approach to the study of the molecular and cellular mechanisms regulating uPAR expression in human airway epithelial cells. It is hoped that a more complete understanding of cell-mediated fibrinolysis will provide the basis for interventions aimed at modulating airway epithelium-dependent lung repair.